The cooperativity phenomena in a pigment-protein complex of light-harvesting antenna revealed by picosecond absorbance difference spectroscopy

A model of the cooperative changes in optical properties of light-harvesting bacteriochlorophyll molecules of complex B890 in response to the absorption of light quanta is proposed. According to the model, each antenna chromophore may persist in either of two optically non-excited states, R and T. The occurrence of at least one excitation per complex causes all optically non-excited chromophores of the complex to be converted from state R to state T. The theory is shown to be in good agreement with experimental ‘light curves’ (ΔAvs intensity of picosecond excitation pulse) for the ‘minor’ and ‘major’ signals of light-harvesting bacteriochlorophylls of complex B890 from Chromatium minutissimum.     

Comparison of the fluorescence kinetics of detergent-solubilized and membrane-reconstituted LH2 complexes from Rps. acidophila and Rb. sphaeroides

Picosecond time-resolved fluorescence spectroscopy has been used in order to compare the fluorescence kinetics of detergent-solubilized and membrane-reconstituted light-harvesting 2 (LH2) complexes from the purple bacteria Rhodopseudomonas (Rps.) acidophila and Rhodobacter (Rb.) sphaeroides. LH2 complexes were reconstituted in phospholipid model membranes at different lipid:protein-ratios and all samples were studied exciting with a wide range of excitation densities. While the detergent-solubilized LH2 complexes from Rps. acidophila showed monoexponential decay kinetics (τ_f = 980 ps) for excitation densities of up to 3·10¹³ photons/(pulse·cm²), the membrane-reconstituted LH2 complexes showed multiexponential kinetics even at low excitation densities and high lipid:protein-ratios. The latter finding indicates an efficient clustering of LH2 complexes in the phospholipid membranes. Similar results were obtained for the LH2 complexes from Rb. sphaeroides. Guest editor: Dr. Conrad Mullineaux.     

Characterization of the core complex of Rubrivivax gelatinosus in a mutant devoid of the LH2 antenna

The core complex of purple bacteria is a supramolecular assembly consisting of an array of light-harvesting LH1 antenna organized around the reaction center. It has been isolated and characterized in this work using a Rubrivivax gelatinosus mutant lacking the peripheral LH2 antenna. The purification did not modify the organization of the complex as shown by comparison with the intact membranes of the mutant. The protein components consisted exclusively of the reaction center, the associated tetraheme cyt c and the LH1 αβ subunits; no other protein which could play the role of pufX could be detected. The complex migrated as a single band in a sucrose gradient, and as a monomer in a native Blue gel electrophoresis. Comparison of its absorbance spectrum with those of the isolated RC and of the LH1 antenna as well as measurements of the bacteriochlorophyll/tetraheme cyt c ratio indicated that the mean number of LH1 subunits per RC-cyt c is near 16. The polypeptides of the LH1 antenna were shown to present several modifications. The α one was formylated at its N-terminal residue and the N-terminal methionine of β was cleaved, as already observed for other Rubrivivax gelatinosus strains. Both modifications occurred possibly by post-translational processing. Furthermore the α polypeptides were heterogeneous, some of them having lost the 15 last residues of their C-terminus. This truncation of the hydrophobic C-terminal extension is similar to that observed previously for the α polypeptide of the Rubrivivax gelatinosus LH2 antenna and is probably due to proteolysis or to instability of this extension.     

On the location of the carotenoids in the light-harvesting pigment-protein complexes of the photosynthetic bacterium Rhodopseudomonas capsulata

Measurements of pronase-induced shifts of the absorption spectrum and of the isobestic point of the light-induced difference spectrum of the carotenoids show that the pool responsible for the light-induced absorption changes in Rhodopseudomonas capsulata wild type is more sensitive to pronase treatment than the bulk carotenoids. The most likely explanation for this, in the context of the work of Kakitani et al. (Kakitani, T., Honig, B. and Crofts, A.R. (1982) Biophys. J. 39, 57–63), is that the field indicating carotenoids, or at least that part of the molecules which determines their spectral characteristics, are imbedded in the LHC II pigment-protein complexes, close to the membrane surface. The importance of the location of the carotenoids for the measurement of the electrical potential differences is briefly discussed.     

Mössbauer spectroscopy studies of photosynthetic reaction centers from Rhodopseudomonas sphaeroides R-26

⁵⁷Fe Mössbauer spectroscopy measurements on reaction centers differing in ubiquinone content, detergent, oxidation state, or the presence of o-phenanthroline all show a single quadrupole doublet of similar splitting (ΔE_Q), center shift (δ) and temperature dependence. The results are indicative of high-spin Fe²⁺ with an approximately invariant first coordination sphere. A crystal field model with strong electron delocalization can account for the temperature dependence of ΔE_Q, but further data are needed to achieve a unique parameterization.     

Shotgun proteomic analysis of a chromatophore-enriched preparation from the purple phototrophic bacterium Rhodopseudomonas palustris

A proteomics approach was evaluated for analysis of photosyntheis-related proteins that are characteristic of chromatophores, particles derived from purple phototrophic bacterial intracytoplasmic membranes. Proteins of purified chromatophores from Rhodopseudomonas palustris were solubilized and digested with trypsin, to create a collection of peptides that were fractionated by liquid chromatography. Peptide sequences were determined and assigned to specific proteins by analysis of tandem mass spectra of peptides, and comparison to a library derived from the recently determined R. palustris genome sequence. A total of 300 proteins were detected with a probability value >/=0.9, and the number of proteins detected increased to 345 when the minimum probability value was reduced to 0.5. Membrane-integral proteins of the reaction center, cytochrome b/c (1), light-harvesting and ATPase complexes were used as controls to assess how well this approach performs with hydrophobic proteins. New genes were identified, and tentatively designated as encoding photosynthesis-related proteins. We conclude that this approach is a powerful method to evaluate the possible existence of new photosynthesis-related proteins (and genes), although alternative methods are needed to evaluate the exact functions of newly discovered genes.     

On the quantitation of bacteriochlorophyll in chromatophore suspensions from purple bacteria

A direct photometric quantitation of bacteriochlorophyll (BChl) at 375 nm in aqueous chromatophore suspensions from various purple bacteria is described. The assay is rapid and reproducible. It is utilized easily for processing large numbers of samples and is as sensitive as extraction methods usually applied today. Drawbacks of extraction methods, particularly not quantitative extractions, photo- and autooxidation are avoided. There is good linearity up to 20 μg BChl/ml suspension, and no interference by buffers is observed.     

Electrostatic effect of surfactant molecules on bacteriochlorophyll a and carotenoid binding sites in the LH1 complex isolated from Rhodospirillum rubrum S1 probed by Stark spectroscopy

The LH1 complexes were isolated from the purple photosynthetic bacterium Rhodospirillum rubrum strain S1. They were initially solubilized using LDAO and then purified in the presence of Triton X-100. The purified complexes were then either used directly or following an exchange into LDAO. Stark spectroscopy was applied to probe the electrostatic field around the bacteriochlorophyll a (BChl a) and carotenoid binding sites in the LH1 complexes surrounded by these two different surfactant molecules. Polarizabilty change () and dipole moment change () upon photoexcitation were determined for the BChl a Q_y band. Both of these parameters show smaller values in the presence of LDAO than in Triton X-100. This indicates that polar detergent molecules, like LDAO, affect the electrostatic environment around BChl a, and modify the nonlinear optical parameters ( and values). The electrostatic field around the BChl a binding site, which is generated by the presence of LDAO, was determined to be |E _L | = ∼3.9 × 10⁵ [V/cm]. Interestingly, this kind of electrostatic effect was not observed for the carotenoid-binding site. The present study demonstrates a unique electrostatic interaction between the polar detergent molecules surrounding the LH1 complex and the Q_y absorption band of BChl a that is bound to the LH1 complex.     

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum

This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.     

Reconstitution of carotenoids into the light-harvesting pigment-protein complex from the carotenoidless mutant of Rhodopseudomonas sphaeroides R26

Two carotenoids, neurosporene and spheroidene, have been successfully added to chromatophores from the carotenoidless mutant of Rhodopseudomonas sphaeroides R26. Carotenoids reconstituted in this way into the B-850 light-harvesting pigment-protein complex both sensitise bacteriochlorophyll fluorescence and protect the complex from the photodynamic reaction.     

Hierarchical organization of the plasma membrane: Investigations by single-molecule tracking vs. fluorescence correlation spectroscopy

Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.     

Triplet Excitation Transfer between Carotenoids in the LH2 Complex from Photosynthetic Bacterium Rhodopseudomonas palustris

We have studied, by means of sub-microsecond time-resolved absorption spectroscopy, the triplet-excited state dynamics of carotenoids (Cars) in the intermediate-light adapted LH2 complex (ML-LH2) from Rhodopseudomonas palustris containing Cars with different numbers of conjugated double bonds. Following pulsed photo-excitation at 590 nm at room temperature, rapid spectral equilibration was observed either as a red shift of the isosbestic wavelength on a time scale of 0.6-1.0 mus, or as a fast decay in the shorter-wavelength side of the T(n)<--T(1) absorption of Cars with a time constant of 0.5-0.8 mus. Two major spectral components assignable to Cars with 11 and 12 conjugated double bonds were identified. The equilibration was not observed in the ML-LH2 at 77 K, or in the LH2 complex from Rhodobacter sphaeroides G1C containing a single type of Car. The unique spectral equilibration was ascribed to temperature-dependent triplet excitation transfer among different Car compositions. The results suggest that Cars of 11 and 12 conjugated bonds, both in close proximity of BChls, may coexist in an alpha,beta-subunit of the ML-LH2 complex.     

Absorbance changes of carotenoids and bacteriochlorophylls responding to the change of local electrical field induced by hydrophobic anion,tetraphenylborate in membranes of photosynthetic bacteria

Large blue-shifts of carotenoid absorption bands were induced by dark addition of a hydrophobic anion, tetraphenylborate, in chromatophores and cell membranes of photosynthetic bacteria, Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata. Tetraphenylborate also induced a red-shift of the 850 nm absorption band and a blue-shift and broadening of the 800 nm band of bacteriochlorophyll. From the analysis of the relation between the magnitude and isosbestic wavelength of the absorbance changes the tetraphenylborate-induced carotenoid band shift were assumed to reflect the change of local electrical field close to each carotenoid molecule which exists as a minor pool on the light-harvesting pigment-protein complex II (LHC II). Absorbance changes of carotenoid and chlorophylls were also induced by tetraphenylborate in membranes of spinach chloroplasts.     

Seven-fold exciton splitting of the 810-nm band in bacteriochlorophyll a-proteins from green photosynthetic bacteria

We report comparative absorbance and fourth derivative absorbance spectra of two different bacteriochlorophyll a-proteins at 5 K in each of two different cryogenic solvent mixtures. In previous studies at 5 K each protein was observed in only one of these mixtures (not the same one). For the protein from Prosthecochloris aestuarii strain 2K, whose structure is known, the solvent effect is relatively small; for the protein from Chlorobium limicola f. sp. thiosulfatophilum strain 6230 (Tassajara), the effect is much more pronounced. From these results together with earlier results at 300 K, we conclude there may be slight conformational differences of the Prosthecochloris protein between the crystalline form used for X-ray diffraction studies and that in a cryogenic solvent. By comparing spectral features of the two proteins in the same solvent, we are able for the first time to assign all seven of the expected exciton levels in each protein. These occur at 793, 801, 806, 810, 814, 819, and 825 nm in the Prosthecochloris protein, and at 793, 802, 806, 810, 816, 820, and 823 nm in the Chlorobium protein.     

Assembly of LH2 light-harvesting complexes in Rhodopseudomonas palustris cells illuminated by blue and red light

We investigated the formation of the B800-850 complex in cells of the bacterium Rhodopseudomonas palustris AB illuminated by red and blue light under anaerobic growth conditions. Under red illumination, the B800-850 complex was assembled with a reduced absorption band at 850 nm. The results of re-electrophoresis of the B800-850 complex and oxidation in the presence of potassium iridate suggest its heterogeneity. It may be a mixture of two complexes (B800 and B800-850). The B800-850 complex lacks the capacity for conformational transitions if assembled under blue illumination. Accordingly, the light-harvesting complex assembled in the blue light contains polypeptides that are not synthesized under normal conditions or at increased or decreased light intensities. The mechanism of regulation of the synthesis of the polypeptides of light-harvesting the B800-850 complex and its dependence on the spectral composition of the light is discussed.     

Low-temperature optical properties and pigment organization of the B875 light-harvesting bacteriochlorophyll-protein complex of purple photosynthetic bacteria

Optical and structural properties of the B875 light-harvesting complex of purple bacteria were examined by measurements of low-temperature circular dichroism (CD) and excitation spectra of fluorescence polarization. In the B875 complex isolated from wild-type Rhodopseudomonas sphaeroides, fluorescence polarization increased steeply across the long-wavelength Q_y bacteriochlorophyll a (BChl) absorption band at both 4 and approx. 300 K. With the native complex in the photosynthetic membranes of Rhodospirillum rubrum and Rps. sphaeroides wild-type and R26-carotenoidless strains, this significant increase in polarization from 0.12 to 0.40 was only observed at low temperature. A polarization of ?0.2 was observed upon excitation in the Q_x BChl band. The results indicate that about 15% of the BChl molecules in the complex absorb at wavelengths about 12 nm longer than the other BChls. All BChls have approximately the same orientation with their Q_y transition dipoles essentially parallel and their Q_x transitions perpendicular to the plane of the membrane. At low temperature, energy transfer to the long-wavelength BChls is irreversible, yielding a high degree of polarization upon direct excitation, whereas at room temperature a partial depolarization of fluorescence by energy transfer between different subunits occurs in the membrane, but not in the isolated complex. CD spectra appear to reflect the two spectral forms of B875 BChl in Rps. sphaeroides membranes. They also reveal structural differences between the complexes of Rps. sphaeroides and Rhs. rubrum, in both BChl and carotenoid regions. The CD spectrum of isolated B875 indicates that the interactions between the BChls but not the carotenoids are altered upon isolation.     

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Q_y transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.     

Effect of monogalactosyldiacylglycerol on the interaction between photosystem II core complex and its antenna complexes in liposomes of thylakoid lipids

The non-bilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant type of lipid in the thylakoid membrane and plays an important role in regulating the structure and function of photosynthetic membrane proteins. In this study, we have reconstituted the isolated major light-harvesting complexes of photosystem II (PSII) (LHCIIb) and a preparation consisting of PSII core complexes and minor LHCII of PSII (PSIICC) into liposomes that consisted of digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), with or without MGDG. Transmission electron microscopy and freeze-fracture studies showed unilamellar proteoliposomes, and demonstrated that most of the MGDG is incorporated into bilayer structures. The impact of MGDG on the functional interaction between LHCIIb and PSIICC was investigated by low temperature (77 K) fluorescence emission spectra and the photochemical activity of PSII. The additional incorporation of LHCIIb into liposomes containing PSIICC markedly increased oxygen evolution of PSIICC. Excitation at 480 nm of chlorophyll (Chl) b in LHCIIb stimulated a characteristic fluorescence emission of the Chl a in PSII (684.2 nm), rather than that of the Chl a in LHCIIb (680 nm) in the LHCIIb–PSIICC proteoliposomes, which indicated that the energy was transferred from LHCIIb to PSIICC in liposome membranes. Increasing the percentage of MGDG in the PSIICC–LHCIIb proteoliposomes enhanced the photochemical activity of PSII, due to a more efficient energy transfer from LHCIIb to PSIICC and, thus, an enlarged antenna cross section of PSII.     

Probing the structure of the core light-harvesting complex (LH1) of Rhodopseudomonas viridis by dissociation and reconstitution methodology

A subunit complex was formed from the core light-harvesting complex (LH1) of bacteriochlorophyll(BChl)-b-containing Rhodopseudomonas viridis. The addition of octyl glucoside to a carotenoid-depleted Rps. viridis membrane preparation resulted in a subunit complex absorbing at 895 nm, which could be quantitatively dissociated to free BChl b and then reassociated to the subunit. When carotenoid was added back, the subunit could be reassociated to LH1 with a 25% yield. Additionally, the Rps. viridis - and -polypeptides were isolated, purified, and then reconstituted with BChl b. They formed a subunit absorbing near 895 nm, similar to the subunit formed by titration of the carotenoid depleted membrane, but did not form an LH1-type complex at 1015 nm. The same results were obtained with the -polypeptide alone and BChl b. Isolated polypeptides were also tested for their interaction with BChl a. They formed subunit and LH1-type complexes similar to those formed using polypeptides isolated from BChl-a-containing bacteria but displayed 6–10 nm smaller red shifts in their long-wavelength absorption maxima. Thus, the larger red shift of BChl-b-containing Rps. viridis is not attributable solely to the protein structure. The -polypeptide of Rps. viridis differed from the other -polypeptides tested in that it could form an LH1-type complex with BChl a in the absence of the - and -polypeptides. It apparently contains the necessary information required to assemble into an LH1-type complex. When the -polypeptide was tested in reconstitution with BChl a and BChl b with the - and -polypeptides, it had no effect; its role remains undetermined.Abbreviations B820 the subunit form of the core light-harvesting complex in BChl-a-containing bacteria which has an absorption maximum at or near 820 nm - B875 the core light-harvesting complex of Rhodobacter sphaeroides which has an absorption maximum at 875 nm - B881 the core light-harvesting complex of wild-type Rhodospirillum rubrum which has an absorption maximum at 881 nm - B895 the subunit form of the core light-harvesting complex in Rps. viridis which has an absorption maximum near 888–895 nm - B1015 the core light-harvesting complex of Rps. viridis which has an absorption maximum at 1015 nm - CD circular dichroism - LH1 the core light-harvesting complex - OG n-octyl -d-glucopyranoside